Generation of human induced pluripotent stem (hiPS) cells from fibroblasts and other somatic cells represents a highly promising strategy to produce auto- and allogenic cell sources for numerous therapeutic approaches as well as novel models of human development and disease2-4. The reprogramming breakthrough1,3,5 involved retroviral transduction of the four factors Oct3/4 (also known as Pou5f1), Sox2, Klf4, and c-Myc in fibroblasts, and since then advances in reprogramming methods have been developed6 using retro- and lenti-viruses transposons9, loxP-flanked lentivirus10, nonintegrating adenoviruses11,12 and plasmids13, proteins14 and RNA15,16. The reprogrammed cells are typically cultured on mouse embryonic fibroblast (MEF) or isogenic human fibroblast feeder layers, and subsequently transferred to feeder layers by mechanical dissociation of pluripotent cell-like colonies for propagation1,3,17. Residual parental or feeder-layer cells introduce experimental variability, pathogenic contamination, and potential immunogenicity18.
iPS cell cultures are often heterogeneous because of the presence of undifferentiated stem cells, non- and partially-induced parental cells and spontaneously differentiated derivatives19. The unavoidable problem of spontaneous differentiation arises from low cell splitting ratios20,21, sub-optimal feeder cultures22, growth factors23, and feeder layer-free substrate quality24. Even under the best of cell culture conditions, some degree of spontaneous differentiation is common and occurs along seemingly random pathways25-29. Spontaneously differentiated (SD)-iPS cells display reduced pluripotency and often contaminate iPS cell cultures, resulting in overgrowth of cultures and compromising the quality of residual pluripotent stem cells23,30,19. The problem of cell contamination is also evident in directed differentiation protocols to generate specific lineages31. For example, differentiation to neural lineages is a step-wise process and intermediate stages like neural rosettes require manual hand-picking because they are contaminated with fibroblast-like cells and residual undifferentiated pluripotent stem cells32,33.
Current methods for propagation of high-quality iPS cell and embryonic stem (hES) cell cultures rely primarily on manual isolation26,27,34-37 alone or in combination with enzymatic dissociation methods. Similar to undifferentiated pluripotent colonies, multi-potent neural rosettes and neurospheres are typically handpicked based on visual inspection and qualitative metrics and transferred for further differentiation into neural progenitors31,32,38. Such methods are tedious, time-intensive, require skilled labor, and are heavily dependent on the ability to morphologically recognize undifferentiated cells. Furthermore, the lack of quality controls affects the reproducibility and consistency of these cultures. Whereas many reagents have been developed for bulk enzymatic passaging, such methods are not selective for iPS cells and therefore unwanted cells are often transferred35,36,39. Furthermore, many enzymatic methods can cause karyotypic abnormalities compared to manual or mechanical passaging34-37. Other technical disadvantages with enzymatic passaging include the need to re-aggregate the dissociated iPS cells as multi-cellular colonies by re-plating on feeder-cells for improved clonal survival20. Although flow cytometry sorting21,23 based on antibody-labeled phenotypic markers can significantly enrich the purity of undifferentiated populations, this method requires single cell dissociation of iPS cells, which induces contractility-mediated programmed cell death40,41, and the plated cells fail to form tightly packed colonies (FIG. 1D). Further, the use of antibody labels is less desirable for therapeutic applications.
Because current techniques for iPS cell purification remain a bottleneck in passaging procedures and suffer from a number of other drawbacks, there is a great need to develop improved technologies that can more efficiently separate colonies of undifferentiated (UD)-iPS cells from contaminating parental cells, feeder cells, or differentiated cells without requiring tedious manual isolation, enzymatic dissociation of iPS cells into single cells and/or labeling with antibodies or other reagents.